Kit and method for detecting the ESM-1 protein

ABSTRACT

The invention concerns a kit for the detection of protein ESM-1 in a sample, comprising:
         a) a first antibody specifically binding to the N-terminal region of protein ESM-1 contained between the amino acid in position 20 and the amino acid in position 78 of the amino acid sequence of this protein; and   b) a second antibody specifically binding to the C-terminal region contained between the amino acid in position 79 and the amino acid in position 184 of the amino acid sequence of protein ESM-1.

This application is a 35 U.S.C. § 371 filing of PCT/FR01/03477, filedNov. 8, 2001, which is based on and claims the benefit of Frenchapplication FR 00/14421, filed Nov. 9, 2000. The entire disclosures ofthese applications are relied upon and incorporated by reference herein.

SCOPE OF THE INVENTION

The present invention concerns a kit for detecting protein ESM-1 in asample. It also relates to a method for detecting protein ESM-1 in asample using such a kit.

The kit and the detection method for protein ESM-1 according to theinvention are industrially applicable particularly for quantification ofprotein ESM-1 in biological samples, and especially biological samplesfrom patients for whom a deterioration of the endothelial vascular wallsis suspected.

STATE OF THE ART

Protein ESM-1 (for “Endothelial-cell-Specific Molecule 1”) is a proteinmainly expressed by the endothelial cells, and predominantly by thevascular endothelial cells. Protein ESM-1 was described for the firsttime by LASSALLE et al. (1996). The messenger RNA of ESM-1 codes for apolypeptide of 184 amino acids whose sequence is described in FIG. 1 ofthe article by LASSALLE et al. (1996).

Protein ESM-1 isolated from the cell lysates of human endothelial cellshas an apparent molecular weight of 20 kDa. The secreted form of proteinESM-1 has an apparent molecular weight of 50 kDa, indicating that thesecreted protein ESM-1 has undergone post-translational modifications.

The use of monoclonal antibodies to detect protein ESM-1 is disclosed inthe French patent application published under the number n^(o)2.775.691and in the article by BECHARD et al. (2000). These monoclonal antibodieswere prepared by immunization of mice with an antigen composed of thesequence running from the amino acid in position 79 up to the amino acidin position 184 of protein ESM-1 which had been fused with protein GST(glutathione-S-transferase). Three families of monoclonal antibodies hadbeen characterized, these antibody families fixing respectively to theantigenic determinants AgD1 79P-99C (antibodies MEP 01, MEP 06 and MEP21), AgD2 119 S-139V (antibodies MEP 08 and MEP13) and AgD3 159G-184R(antibodies MEP 04, MEP 14 and MEP19).

BECHARD et al. (2000) described the development of an immunoenzymatictest of the “sandwich” type using the monoclonal antibodies MEP 19 andMEP 21 to perform a test for detecting protein ESM-1 in a sample. Thistest allowed the authors to demonstrate strong production of proteinESM-1 in the serum of patients hospitalized for septic shock. Theresults presented in FIG. 4 of this article show that the detection testusing the antibodies MEP 19 and MEP 21 enables a concentration ofprotein ESM-1 above 4 nanograms per milliliter to be detected.

However, although the immunoenzymatic test for protein ESM-1 describedby BECHARD et al. (2000) is suitable for high concentrations of proteinESM-1 in a sample, it does not detect low quantities of this protein,which are however significant in physiological disorders, such as adeterioration of the endothelial cell wall in patients, as is shownaccording to the invention.

SUMMARY OF THE INVENTION

The applicant has endeavoured to develop an immunological test for thedetection of protein ESM-1 in a sample which is much more sensitive thanthe test described in the state of the art.

The object of the invention is a kit for the detection of protein ESM-1in a sample, comprising:

a) a first antibody facing specifically to the N-terminal regioncontained between the amino acid in position 1 and the amino acid inposition 78 of the amino acid sequence of protein ESM-1; and

b) a second antibody fixing specifically to the C-terminal regioncontained between the amino acid in position 79 and the amino acid inposition 184 of the amino acid sequence of protein ESM-1.

A further object of the invention is a method for detecting proteinESM-1 in a sample, characterized in that it comprises the followingsteps:

a) placing the sample to be tested in contact with a support on which isimmobilized a first antibody fixing specifically to the N-terminalregion or the C-terminal region of the amino acid sequence of proteinESM-1;

b) placing the complex potentially formed between the immobilizedantibody and protein ESM-1 in contact with a second antibody fixingspecifically to the N-terminal region or the C-terminal region notrecognized by the first antibody; and

c) detecting the complex potentially formed between protein ESM-1 andthe second antibody.

The invention also concerns certain antibodies used in the above method.

A further object of the invention is the use of the detection kitaccording to the invention for detecting a deterioration of theendothelial vascular wall in a patient, particularly in a patientsuffering from septic shock or cancer or a patient undergoing atherapeutic treatment likely to be cytotoxic against vascularendothelial cells.

DETAILED DESCRIPTION OF THE INVENTION Detection Kit According to theInvention

The detection kit according to the invention enables the presence ofprotein ESM-1 to be detected in a sample with high sensitivity.

The sample in which the presence of protein ESM-1 is sought may be ofany type. It is preferably a biological fluid such as serum, plasma,whole blood, urine, cerebrospinal fluid, or culture supernatants or celllysates.

The applicant has shown that protein ESM-1 secreted by eukaryotic cellshas specific properties which are not found in protein ESM1 produced byprokaryotic cells, such as cells of Escherichia coli.

Protein ESM-1 is secreted by eukaryotic cells, and particularly byendothelial cells, in the form of a glycoprotein, more precisely aproteoglycan having a chain of the chondroitin/d rmatan sulfat type. Inaddition, the applicant has shown that protein ESM-1 secreted byeukaryotic cells undergoes other post-translational modificationsincluding conformational modifications caused by the formation ofdisulfide bridges between the cysteine residues of the N-terminal regionof this protein. The polypeptide ESM-1 of 184 amino acids is referencedas sequence SEQ ID N^(o)1.

In order to develop a highly sensitive immunological detection test forprotein ESM-1, the applicant has pursued the strategy of preparingmonoclonal antibodies directed against the N-terminal region runningfrom the amino acid in position 20 to the amino acid in position 78 ofthe sequence SEQ ID N^(o)1 described by LASSALLE et al. (1996), theamino acid in position 20 constituting the amino-terminal amino acid ofthe secreted protein ESM-1.

The monoclonal antibodies prepared by the applicant are preferablydirected against the protein ESM-1 secreted by eukaryotic cells andhaving undergone the post-translational modifications mentioned above.

More specifically, the applicant has produced cell lines of hybridomasproducing monoclonal antibodies fixing specifically to the region ofprotein ESM-1 undergoing conformational modifications arising from thecreation of disulfide bridges, in other words antibodies directedagainst the N-terminal region of protein ESM-1 running from the aminoacid in position 20 up to the amino acid in position 78 of the aminoacid sequence of protein ESM-1 of sequence SEQ ID N^(o)1.

In order to obtain a tool for detecting protein ESM-1 in a sample, theapplicant has developed an immunological test of the “sandwich” typeusing two antibodies specifically binding to two distinct regions ofprotein ESM-1, respectively an antibody binding specifically to theN-terminal region running from the amino acid in position 20 up to theamino acid in position 78 of ESM-1 and an antibody binding specificallyto the C-terminal region running from position 79 to position 184 of thesequence of ESM-1.

A first object of the invention thus consists of a kit for detectingprotein ESM-1 in a sample, said kit comprising:

a) a first antibody binding specifically to the N-terminal region ofprotein ESM-1 contained between the amino acid in position 20 and theamino acid in position 78 of the amino acid sequence of this protein;and

b) a second antibody specifically binding to the C-terminal regioncontained between the amino acid in position 79 and the amino acid inposition 184 of the amino acid sequence of protein ESM-1.

The antibody binding specifically to the N-terminal region of ESM-1 ispreferably directed specifically against the N-terminal region ofprotein ESM-1 secreted by eukaryotic cells and having undergone thepost-translational modifications detailed above. This means that theantibody binding specifically to the N-terminal region has been obtainedaccording to a method including a step in which an animal has beeninjected with a purified preparation of protein ESM-1 produced by aeukaryotic cell.

“Antibody,” in the context of the invention, should be understood to bea molecule containing a “paratope” able to bind specifically to theN-terminal region or to the C-terminal region of protein ESM-1.“Antibody,” according to the invention, should be understood also as ahomogeneous population of molecules all containing the same “paratope”able to bind specifically to the N-terminal region or to the C-terminalregion of protein ESM-1.

“Paratope” should be understood as the antigenic combination sitecontained in the Fab fragment of an antibody, which is localized in thehypervariable or CDR domains of the variable domains V_(H) and V_(L) ofthe heavy chain or the light chain of an immunoglobulin.

An antibody according to the invention may be prepared from hybridomasaccording to the technique described by KOHLER and MIELSTEIN (1975), orby the technique of the hybridoma of human B cells described by KOZBOR(1983). An antibody according to the invention also includes thefragments of single chain Fv chimeric antibodies (ScFv for “Single ChainFv”) such as disclosed in the U.S. Pat. No. 64.946.778 and by MARTINEAUet al. (1998). An antibody according to the invention may also beproduced by the phage banks (“Phage Display Libraries”) such as thosedescribed by RIDDER et al. (1995). An antibody according to theinvention may also be a human antibody produced according to thetechnique described by REINMANN et al. (1997) or by the techniquedescribed by LEGER et al. (1997).

The detection kit for protein ESM-1 according to the inventionpreferably comprises one of the two antibodies immobilized on a support.In this preferred embodiment, the detection kit is presented in a “readyto use” form to perform an immunodetection test of the “sandwich” typefor protein ESM-1.

It has been shown according to the invention that the use of a kit suchas defined above for the detection of the presence of protein ESM-1 in asample results in a detection sensitivity 10 times greater than thatobserved with the test for detection of ESM-1 described by BECHARD etal. (2000).

The high sensitivity of the immunodetection test according to theinvention has made it possible to detect very low concentrations ofprotein ESM-1 in a biological sample, for example in human serum, andhas thus allowed early detection of the deterioration of the endothelialvascular walls in a patient.

In particular, the use of an immunodetection kit according to theinvention has enabled serum concentrations of ESM-1 of from 1 to 3nanograms per ml to be detected in “atopic” individuals suffering fromsepsis.

As is shown in the examples, the use of an immunodetection kit accordingto the invention can be used to detect concentrations of ESM-1 of theorder of 100 to 200 picogram/ml, while the test described by BECHARD etal. (2000) does not detect concentrations of ESM-1 of less than 1nanogram/ml.

The antibody directed against the N-terminal region of ESM-1 isadvantageously a monoclonal antibody produced by a hybridoma lineobtained after immunization of a mammal, preferably a mouse, withrecombinant protein ESM-1 synthesized by a eukaryotic cell, for examplea cell of the CHO line transformed by an expression vector containing aDNA insert coding for protein ESM-1 described by LASSALLE et al., forexample the vector pcDNA3.

It is preferably the monoclonal antibody produced by the hybridoma linedesignated MEC 15 deposited at the Collection de Cultures deMicro-organismes (CNCM) of the Institut Pasteur, located at InstitutPasteur, 28 rue du Docteur Roux, F-75724 Paris Cedex 15, France, on 17Oct. 2000 under the access number I-2572.

The MEC15 antibody constitutes an object of the invention.

The antibody binding specifically to the C-terminal part of the proteinESM-1 may be either an antibody directed against protein ESM-1 producedby a prokaryotic cell, such as Escherichia coli, or by a eukaryoticcell, such as cells of the CHO line.

The antibody binding specifically to the C-terminal part of the proteinESM-1 is preferably chosen from among the antibodies able to recognizeone of the three following antigenic determinants of protein ESM-1:

-   -   the determinant AgD1 running from the proline residue in        position 79 up to the cysteine residue in position 99 of ESM-1,        such as the antibodies produced by the hybridoma lines MEP01,        MEP06 and MEP21 described by BECHARD et al. (2000);    -   the antigenic determinant AgD2 running from the serine residue        in position 119 up to the valine residue in position 139 of        ESM-1 recognized by the antibodies produced by the hybridomas        MEP08 and MEP13 described by BECHARD et al. (2000);    -   the antigenic determinant AgD3 running from the glycine residue        in position 159 up to the arginine residue in position 184 of        ESM-1 is recognized by the antibodies produced by the hybridoma        lines MEP04, MEP14 and MEP19 described by BECHARD et al. (2000).

In a preferred embodiment, a person skilled in the art mayadvantageously refer, for the use of an antibody specifically binding tothe C-terminal region of protein ESM-1, to the following antibodies:

-   -   the specific monoclonal antibodies of the antigenic determinant        D1 produced by the hybridoma line deposited at the CNCM on 19        Nov. 1997 under the access number I-1944 (antibody MEP 21);    -   the specific monoclonal antibodies of the antigenic determinant        D2 produced by the hybridoma line deposited at the CNCM on 19        Nov. 1997 under the access number I-1941 (antibody MEP08);    -   the specific monoclonal antibodies of the antigenic determinant        D3 produced by the hybridoma line deposited at the CNCM on 19        Nov. 1997 under the access numbers I-1942 (antibody MEP14) and        I-1943 (antibody MEP19).

In the preferred embodiment of the immunodetection kit according to theinvention, the first and the second antibodies are chosen so that theirrespective recognition sites on the protein ESM-1 are very distant fromeach other in order to avoid any occurrence of competitive fixation ofone antibody with respect to the other on the protein, which could becaused by a steric hindrance phenomenon in the case where the respectiverecognition sites of the two antibodies are too close.

Thus, in a preferred embodiment of a detection kit according to theinvention, the first antibody specifically binding to the N-terminalregion of protein ESM-1 is the antibody produced by the hybridoma lineMEC15. According to this preferred embodiment, the antibody specificallybinding to the C-terminal region of protein ESM-1 is the monoclonalantibody produced by the hybridoma line MEP14.

In general, an antibody directed against the C-terminal region ofprotein ESM-1 may be selected according to the technique described byBECHARD et al. (2000), which consists of preparing an expression vectorcontaining a DNA insert coding for the C-terminal region of proteinESM-1 fused to the protein GST, then to immunize mice with purifiedprotein ESM-1 obtained from cell lysates of Escherichia coli cellstransformed with the said expression vector. After fusion of mousespleen cells thus immunized with myeloma cells in order to obtain aseries of hybridomas producing monoclonal antibodies, the antibodies ofinterest binding specifically to the C-terminal region of protein ESM-1may be selected by mapping the epitopes using peptides derived fromESM-1 containing progressive deletions of the N-terminal end of theC-terminal region of this protein.

Similarly, a skilled person may select the antibodies specificallybinding to the N-terminal region of protein ESM-1 by immunizing micewith whole protein ESM-1, preferably whole protein ESM-1 produced by aeukaryotic host cell, then by producing a series of hybridoma lines fromthe spleen cells of the immunized mice, then by selecting the antibodiesof interest specifically binding to the N-terminal region, for example,by competitive immunodetection tests with the antibodies specificallyfixing to the C-terminal region of ESM-1. The antibodies of interest arethose that do not compete with the specific antibodies of the C-terminalregion of ESM-1, in other words the antibodies that do not compete withthe specific antibodies of the antigenic determinants D1, D2, and D3 ofprotein ESM-1.

One of the two antibodies comprising the detection kit according to theinvention is preferably immobilized on a support. The support on whichthe antibody is immobilized may be of any type known to a person skilledin the art specializing in immunodetection tests, in particular inimmunodetection tests of the “sandwich” type.

As an illustration, the support on which the antibody is immobilized maybe a porous or non-porous material insoluble in water. The support maybe hydrophilic and contain inorganic powders such as silica, magnesiumand aluminium sulfate, natural polymers, in particular cellulosematerials and their derivatives, natural or synthetic polymers such asnitro-cellulose, cellulose acetate, poly(vinyl chloride),polyacrylamide, crosslinked dextran, agarose, polyacrylate,polyethylene, polypropylene, poly(4-methylbutene), polystyrene,polymethacrylate, poly(ethylene terephthalate), Nylon, poly(vinylbutyrate), certain types of glass such as Bioglass, or ceramics.

The fixation of an antibody according to the invention onto a supportmay be performed by techniques well known to a skilled person. Thesupport may be in various different forms, including strips or inparticular beads. The surface of the support may be polyfunctional orable to be polyfunctionalized so as to fix the antibody via covalent ornon-covalent interactions which may be specific or non-specific. As anillustration, for the immobilization of an antibody onto a support, aperson skilled in the art may advantageously refer to the U.S. Pat. No.4,168,146 or the U.S. Pat. No. 4,347,311.

One of the antibodies comprising the detection kit is advantageouslycovalently bound to a molecule enabling its direct or indirectdetection.

In the embodiment in which the antibody is immobilized on a support, theother antibody is preferably covalently bound to a molecule enabling itsdirect or indirect detection.

The detectable molecule may be isotopic or non-isotopic.

As an illustrative but non-limiting example, the detectable molecule maybe involved in a catalytic reaction, such as an enzyme, an enzymefragment, an enzyme substrate, an enzyme inhibitor, a coenzyme or acatalyst. The detectable molecule may also be a chromogen, such as afluorophore, a colorant, or a chemiluminescent molecule.

The detectable molecule may thus be a fluorescent molecule such as themolecules described by ICHINOSE at al. (1991) or the fluorescentisothiocyanate derivatives, phycoerithrine, rhodamine isothiocyanate,dansyl chloride or the compound XRITC, protein GFP (“Green FluorescentProtein”) of the fish Aequorea Victoria and its many derivatives, orprotein YFP (“Yellow Fluorescent Protein”) as well as the proteinluciferase.

Among the detectable molecules with catalytic activity, the preferredmolecules are the following enzymes, according to the InternationalClassification I.U.B.: (i) the oxidoreductases of class 1 and (ii) thehydrolases of class 3. The preferred oxidoreductases are (i) thedehydrogenases of class 1.1, most particularly 1.1.1, 1.1.3 and 1.1.99;(ii) the peroxidases of class 1.11; and (iii) the hydrolases of class3.1, most particularly of class 3.1.3 and class 3.2, most particularly3.2.1. The preferred dehydrogenases are malate dehydrogenase,glucose-6-phosphate dehydrogenase and lactate dehydrogenase. Thepreferred oxidase is glucose oxidase. The preferred peroxidase ishorseradish peroxidase. The preferred hydrolases are the alkalinephosphatases, β-lucosidase and lyzozyme.

The detectable molecule may also be a radioactively marked molecule, forexample by an isotope chosen from among [³H], [³²P] and [¹²⁵I].

In the embodiment in which the detectable molecule comprises an indirectmarker, one of the antibodies comprising the detection kit according tothe invention may be covalently bound to a ligand such as biotin orstreptavidine.

In this particular embodiment, the detectable molecule is chosen so thatit fixes onto the ligand which is covalently bound to the antibody. Thedetectabl molecule may for example be itself bound respectively tobiotin or to streptavidine.

According to another embodiment of a detection kit according to theinvention, the means of revealing the formation of a complex between theprotein ESM-1 present in the sample tested and one of the specificantibodies of protein ESM-1, may be an antibody, for example an antibodyable to bind specifically to the Fc part of the anti-ESM-1 antibody oran antibody able to bind specifically to the isotype to which theanti-ESM-1 antibody belongs, for example an antibody specificallyrecognizing the mouse antibody of the isotype IgG1.

In a preferred embodiment of a detection kit according to the invention,it is the antibody specifically recognizing the C-terminal region ofprotein ESM-1 which is immobilized on a support.

Such a detection kit is advantageously that in which the antibodyproduced by the hybridoma MEP14 (CNCM N^(o)I-1942) is immobilized on thesupport, is the antibody specifically binding to the N-terminal regionof protein ESM-1 and the antibody produced by the hybridoma MEC15 (CNCMN^(o)I-2672).

The detection tool may thus be a biotinylated monoclonal antibody, forexample from a rat, specifically recognizing the mouse antibody of theisotype IgG1, the detection system being supplied by astreptavidine-peroxidase conjugate.

Immunodetection Method According to the Invention

A further object of the invention is a method for detecting proteinESM-1 in a sample characterized in that it comprises the following steps

a) placing the sample to be tested in contact with a first antibodyspecifically binding to the N-terminal region of protein ESM-1 or with afirst antibody binding to the C-terminal region of protein ESM-1.

b) placing the complex potentially formed between protein ESM-1 presentin the sample and the first antibody in contact with a second antibodyspecifically binding to the region of protein ESM-1 chosen from amongthe N-terminal region and the C-terminal region not recognized by thefirst antibody; and

c) detecting the complex formed between protein ESM-1 and the secondantibody

By “complex formed between protein ESM-1 and the second antibody” shouldbe understood the complex formed between the complex formed between:

-   -   the complex formed between protein ESM-1 and the first antibody,        and    -   the second antibody.        According to a first embodiment of the immunodetection method        above, the first or the second antibody may be immobilized on        the surface of a support.

According to a second embodiment of the immunodetection method above,the antibody which is immobilized on a support is an antibodyspecifically binding to the C-terminal region of protein ESM-1 and thesecond antibody is an antibody specifically binding to the N-terminalregion of this protein.

According to a third embodiment of the immunodetection method above, theantibody which is immobilized on a support is an antibody specificallybinding to the N-terminal region of protein ESM-1 and the secondantibody is an antibody specifically binding to the C-terminal region ofthis protein.

The antibodies specifically binding onto the N-terminal or C-terminalregions of ESM-1 are as defined above.

In a preferred embodiment of the detection method according to theinvention, the antibody specifically binding to the N-terminal region ofprotein ESM-1 is the antibody MEC15 produced by the hybridoma depositedon 17 Oct. 2000 at the CNCM under the access number I-2572.

The second antibody may be the antibody MEP14 produced by the hybridomadeposited at the CNCM on 19 Nov. 1997 under the access number I-1942.

Step c) of the method described above may be performed using abiotinylated antibody able to fix to a second antibody, the detectablemolecule being composed of a conjugate of the streptavidine-peroxidasetype.

Application of the Detection Method According to the Invention

The applicant has shown that the method of immunodetection of proteinESM-1 according to the invention enables the serum concentration ofprotein ESM-1 in humans to be quantified with high sensitivity, inparticular in patients suffering from sepsis, and enables a correlationto be established between the quantity of protein ESM-1 circulating andthe severity of the sepsis in the patients.

According to the standard defined by the American College of ChestPhysicians/Society of Critical Care Medicine Consensus Conference (1992,definitions for sepsis and multiple organ failure, and guidelines forthe use of innovative therapies in sepsis; Crit Care Med., vol. 20:864-874), sepsis may be divided into three categories of increasingseverity: sepsis, severe sepsis and septic shock.

Thanks to the immunodetection method according to the invention, acorrelation has been established between the severity of the sepsis andthe concentration of protein ESM-1 circulating in the patients.

In addition, the high sensitivity of the immunodetection test accordingto the invention has shown that patients suffering from simple sepsishad a concentration of circulating protein ESM-1 which, although low,was significantly detectable and significantly higher (p<0.001) than theconcentration of protein ESM-1 found in the serum of healthy volunteers.

Up until now, the development of sepsis in a patient was monitored bythe quantification of three markers, respectively C-reactive protein,soluble ICAM-1 protein and procalcitonin.

It has been shown according to the invention that the development of thelevels of C-reactive protein and of soluble ICAM-1 protein do notcorrelate with the development of the concentration of ESM-1.

In contrast, there is a good correlation between the development of thelevels of procalcitonin and of protein ESM-1. However, the biologicalsignificance of procalcitonin in the case of sepsis is not known andthis protein therefore does not represent a good biological marker forthe development of sepsis.

On the other hand, protein ESM-1, due to its role in the regulation ofinflammatory reactions, constitutes a new marker of the development ofsepsis whose physiological significance is directly linked to theuncontrolled development of the inflammatory reaction, particularly therecruitment of leukocytes during the phenomena of extravasation andmassive infiltration of these cells in different tissues, especiallylung tissue, which are causal, or at the least concomitant, phenomena,with a deterioration of the endothelial vascular wall.

The applicant has thus shown that the immunodetection test of theinvention enables low concentrations of circulating protein ESM-1 to bedetected, of the order of 1 to 3 nanogram per milliliter, these lowconcentrations found in atopic patients may be dearly distinguished fromthe base concentrations of the order of nanogram/ml or even lowermeasured in healthy volunteers.

In addition, it has been shown that the quantity of serum ESM-1 found inpatients suffering from sepsis represents a reliable diagnosis ofmortality for these patients. Thus, for 68 patients tested, the patientshaving died 10 days after their hospitalization presented concentrationsof circulating ESM-1 at day 0 and at day 1 significantly higher than theconcentrations of ESM-1 found in the serum of patients having survived(10.6±0.8 nanograms per ml in deceased patients against 4.0±0.6nanograms per ml in patients having survived [p>0.01]).

This close correlation between the level of circulating protein ESM-1and the prognosis of mortality of the patients has not been found forthe conventional markers of sepsis, in other words C-reactive protein,procalcitonin and soluble ICAM-1 protein.

However, the measurement of the concentration of circulating ESM-1 alonein a patient is not sufficient for performing a medical diagnosispermitting the doctor to take therapeutic measures suitable for theoverall state of the patient.

A further object of the invention consists of the use of a detection kitfor protein ESM-1 according to the invention to detect in vitrodeteriorations of the endothelial vascular wall in man, and particularlyin patients.

The invention also relates to the use of a detection kit for proteinESM-1 as defined above for monitoring in vitro a marker of the severityof a sepsis in a patient.

It has also been shown according to the invention that a concentrationof circulating protein ESM-1 higher than normal is found in patientshaving undergone organ transplants and treated with an immunosuppressantcompound. The increase in the concentration of circulating protein ESM-1is indicative of a cytotoxic activity of the immunosuppressant compoundagainst endothelial cells, particularly endothelial cells of thevascular wall.

Thus, the use of a detection kit for protein ESM-1 according to theinvention makes it possible to monitor patients treated withimmunosuppressant compounds and to determine the time when theseimmunosuppressants become cytotoxic.

The detection of levels of circulating ESM-1 higher than normal in thesepatients may represent an indication for the doctor to moderate thedoses of immunosuppressant compounds administered, when this measurementis accompanied by other physiological parameters in the patient.

According to a further aspect, the invention concerns the use of animmunodetection kit according to the invention for the quantification ofprotein ESM-1 in vitro in a patient treated with an immunosuppressantcompound, in particular in a patient having undergone an organtransplant.

The applicant has also shown a significant increase of the level ofcirculating protein ESM-1 in patients suffering from cancer, inparticular broncho-pulmonary cancer.

According to yet another aspect, the invention concerns the use of animmunodetection kit such as defined above for the quantification invitro of protein ESM-1 in a patient suffering from cancer.

In general, a level of circulating protein ESM-1 higher than 1 nanogramper ml is significant of a deterioration of the endothelial cells of thevascular wall and may thus represent a parameter to be taken intoaccount during the establishment of a clinical diagnosis of a patient bya doctor, it being understood that this parameter alone, taken inisolation, cannot in itself establish a therapeutic or clinicaldiagnosis for a patient.

The present invention is in addition illustrated, without in any waybeing limited, by the following figures and examples.

FIGURES

FIG. 1 illustrates a comparison of the sensitivity of detection of theimmunodetection test for protein ESM-1 according to the invention (opencircles) with the detection test described by BECHARD et al. (2000)represented by solid circles. The ordinate represents the concentrationof protein ESM-1 in the sample to be tested, expressed in nanograms perml. The optical density is represented on the abscissa.

FIG. 2 illustrates the immunodetection profiles performed according tothe immunoenzymatic technique of the “sandwich” type. In all cases, themonoclonal antibody produced by the hybridoma MEP14 was adsorbed on amicro-titration plate. The optical density is represented on theabscissa. The ordinate represents the concentration in protein ESM-1produced by the cells of the line HEK293, expressed in nanograms per ml.The second antibody used in the immunodetection test was an antibodydirected specifically against the N-terminal region of protein ESM-1. Itwas antibody MEC2 (FIG. 2A), MEC15 (FIG. 2B) and MEC36 (FIG. 2C).

FIG. 3 illustrates a comparison of the concentration of protein ESM-1found in the plasma or the serum of healthy volunteers or of patients.The ordinate represents the serum concentration of protein ESM-1,expressed in nanograms per ml. the abscissa represents the plasmaconcentration of protein ESM-1, expressed in nanograms per ml.

FIG. 4 illustrates the quantity of circulating protein ESM-1 found inthe serum of healthy volunteers or of patients suffering from sepsis ofincreasing clinical severity. The concentration of serum protein ESM-1,expressed in nanograms per ml, is shown on the ordinate. The abscissashows the different populations of individuals tested, respectivelyhealthy subjects (CTRL), patients suffering from sepsis, severe sepsisor septic shock, according to the clinical definition published in Crit.Care Med., 1992, vol. 20:864-874.

FIG. 5 illustrates the quantification of three sepsis markers inpopulations of healthy patients, or of patients suffering from sepsis,severe sepsis or septic shock.

FIG. 5A illustrates the quantification of soluble ICAM-1 protein in theserum, expressed in nanograms per ml.

FIG. 5B illustrates the quantification of C-reactive protein, expressedin milligrams per ml.

FIG. 5C illustrates the quantification of serum procalcitonin, expressedin nanograms per ml.

FIG. 6 illustrates a comparison between the levels of circulatingprotein ESM-1, and of each of the three conventional sepsis markers,respectively soluble ICAM-1 protein, C-reactive protein and Iaprocalcitonin at day 0 of hospitalization of the patients, respectivelyin patients having survived and patients having died.

FIG. 6A illustrates the serum level of protein ESM-1, expressed innanograms per ml.

FIG. 6B illustrates the serum level of soluble ICAM-1 protein, expressedin nanograms per ml.

FIG. 6C illustrates the serum level of C-reactive protein, expressed innanograms per ml.

FIG. 6D illustrates the level of procalcitonin, expressed in nanogramsper ml.

FIG. 7 illustrates the prognostic value of the quantity of protein ESM-1or of soluble ICAM-1 protein in patients at day 0 and at day 1 afterhospitalization, compared to their mortality.

FIG. 7A illustrates the level of protein ESM-1, expressed in nanogramsper ml, found respectively in patients having survived (solid circle)and patients having died (open circle), at day 0 and day 1 afterhospitalization.

FIG. 7B illustrates the level of serum soluble ICAM-1 protein in thesame patients.

FIG. 8 illustrates the mortality prognostic value of the level ESM-1 andsoluble ICAM-1 protein measured at day 0 and at day 1 afterhospitalization.

FIG. 8A illustrates the level of serum protein, expressed in nanogramsper ml, in patients having survived (solid circle) and patients havingdied (open circle).

FIG. 8B illustrates the level of serum soluble ICAM-1 protein, expressedin nanograms per ml, in patients having survived (solid circle) andpatients having died (open circle)).

EXAMPLES Example 1 Preparation of Monoclonal Antibodies SpecificallyBinding to the N-terminal Region of Protein ESM-1 Produced by EukaryoticCells

In order to obtain anti-ESM-1 monoclonal antibodies directed against theN-terminal region of protein ESM-1 rich in cysteine residues, the nativeform of protein ESM-1 produced by a CHO cell line transfected by anexpression vector containing a DNA insert coding for protein ESM-1 waspurified.

The cDNA sequence coding for protein ESM-1 is referenced as sequence SEQID N^(o)2 in the list of sequences.

The cDNA of ESM-1 was inserted into the eukaryotic expression vectorpcDNA3 (In vitrogen) then transfected in CHO cells with lipofectamine(Gibco) according to the recommendations of the manufacturer. 48 h afterthe transfection the cells were subcultured in the presence of aselection agent (G418, Gibco) at a dose of 1000 micrograms/ml). Aftertwo weeks of selection the CHO cells resistant to G418 were cloned bylimiting dilution. The clones expressing ESM-1 were then selected andnamed CHO-ESM (deposited at the CNCM).

For the production, the CHO-ESM cells were cultured in suspension in amedium without foetal calf serum (medium CHO SFM II, Gibco). Thesupernatant was adjusted to pH 8 and passed over a column ofDEAE-sepharose (Pharmacia). The column was washed with a buffer 50 mMTris, pH 8, 0.2 M NaCl. The pmolecule ESM-1 was eluted in a buffer 50 mMTris, pH 8, 1 M NaCl. The eluate was then diluted 1:4 in a buffer 50 mMTris, pH 8 and incubated in the presence of anti-ESM-1 monoclonalantibody (MEC4) immobilized on agarose (Biorad). After one night ofincubation at 4° C. with agitation, the agarose beads were washed withthe buffer 50 mM Tris, pH 8, 0.2 M NaCl. ESM-1 was eluted with 3 MMgCl2, the eluate was concentrated and dialysed in the buffer 50 mMTris, pH 8, 0.5 M NaCl and stored at −70° C.

Balb/C mice were immunized by injection of 10 μg of purified recombinantprotein ESM-1 per mouse, according to a standard immunization protocolin the presence of Freund's adjuvant.

Hybridoma cells secreting anti-ESM-1 monoclonal antibodies were obtainedby fusion, screening and sub-cloning according to the techniquedescribed by BECHARD et al. (2000).

Five hybridoma cell clones were obtained and were generically designatedMEC (“Mouse Monoclonal Antibody to ESM-1 produced by CHO Cells”).

Four of the hybridomas selected were of isotype IgG1,k respectively thehybridomas designated MEC4, MEC5, MEC15 and MEC36.

One of the hybridomas was of isotype IgM,k, the hybridoma MEC11.

The hybridoma cell clones were cultured in a culture medium in theabsence of serum and the anti-ESM-1 antibodies were purified bychromatography on a column of protein G-Sepharose marketed by Pharmacia(UPPSALA, Sweden).

Example 2 Selection of the Monoclonal Antibodies Specifically DirectedAgainst the N-terminal Part of Protein ESM-1 Produced by Cells of theCHO Line

The MEC antibodies of class IgG1,K were selected by the persistence of astrong binding between the antibodies of the MEC series and ESM/Fc inthe presence of MEP antibody of different isotype and at a finalconcentration of 1 μg/ml. The tests were performed by ELISA according tothe examples above for the selection of the MEP and MEC antibodies(particularly by the immuno-detection by competition experimentsdescribed by LASSALLE et al. (1996).

Example 3 Immuno-detection Test for Protein ESM-1 According to theInvention

The immuno-detection test consisted of an immuno-enzymatic test of the“sandwich” type whose overall characteristics were identical to thatdescribed by BECHARD et al. (2000).

The anti-ESM-1 monoclonal antibody produced by the hybridoma line MEP14(CNCM N^(o)I-1942) was diluted to a concentration of 5 μg/ml in a buffercarbonate 0.1 M, pH 9.5, and adsorbed overnight at +4° C. on a 96-wellplate (plate E.I.A./R.I.A., Costar, Cambridge, Mass., USA).

The plate was saturated for one hour at laboratory temperature with avolume of 200 μl/well of PBS buffer containing 0.1% of bovine serumalbumin and 5 mM of EDTA, then washed twice with an ELISA buffer (thePBS buffer above supplemented with 0.1% of Tween 20).

A calibration was performed with purified protein ESM-1 according to thetechnique described by BECHARD et al. (2000).

Blood samples were serially diluted (1:2 to 1:128), in an ELISA bufferand incubated on an ELISA plate for one hour at laboratory temperature.

The wells were washed three times with an ELISA buffer then incubatedfor 1 hour at laboratory temperature with a second monoclonal antibodydirected against ESM-1, the antibody MEC15 (CNCM N^(o)I-2572) at aconcentration of 0.1 μg/ml in 100 μl of buffer per well.

After three washings, a biotinylated rat monoclonal antibody directedagainst mouse IgG1 (marketed by PHARMINGEN) diluted in an ELISA bufferwas added and left to incubate for one hour.

After three washings in ELISA buffer, the wells were incubated with astreptavidine-peroxidase conjugate at a dilution 1:10.000 v/v (marketedby ZYMED).

After 30 minutes of incubation with the streptavidine-peroxidaseconjugate, three washings of each well were performed in an ELISAbuffer, then two washings in a PBS buffer.

The streptavidine-peroxidase conjugate was revealed with the substrateTMB marketed by SIGMA (Saint-Louis, Mo., USA) in the presence of 5 μl ofH₂O₂ for 30′.

The revelation reaction was stopped by addition of a volume of 100 μl ofH₂SO₄ 2N.

The plate was read using a spectrophotometer (anthos labtec LP40.France) at a wavelength of 405 nanometers.

The plasma or serum concentration of protein ESM-1 was calculated fromthe optical density measurements and expressed in nanograms per ml.

Example 4 Comparative Results of the Determination of ESM-1 with theImmuno-detection Test According to the Invention and an Immuno-detectionTest of the State of the Art

Comparative determinations of known concentrations of recombinantprotein ESM-1 produced by the transfected CHO cell line, and thenpurified as described in example 1, were performed.

The first determination was performed in accordance with the teaching ofexample 3. Briefly, the antibody MEP14 was immobilized on a 96-wellplate of the ELISA type, then placed in contact with a series of PBSbuffer solutions containing a known concentration of protein ESM-1.After washing, the complex formed between the immobilized antibody MEP14and protein ESM-1 was incubated with a buffer solution containing theantibody MEC15 of the invention.

After a further series of washings, the antigen/antibody complex formedwas incubated successively with a biotinylated rat anti-IgG1 mouse, thenwith a streptavidine-peroxidase conjugate before revelation withhydrogen peroxide.

The second test was performed under the same conditions, but using theantibody MEP19 immobilized on a 96-well plate of the ELISA type and theantibody MEP21 as the second antibody in this immunoenzymatic techniqueof the “sandwich” type.

The results are given in FIG. 1.

With the combination of antibodies according to the invention, asignificant difference of the optical density value was obtained with aconcentration of protein ESM-1 of the order of 0.15 to 0.2 nanograms permilliliter.

In contrast, with the combination of the antibodies MEP19 and MEP21, asignificant increase in the optical density value was only obtained fora concentration of ESM-1 equal to 1 or 2 nanograms per ml.

The results given in FIG. 1 clearly show the high sensitivity of theimmunodetection test for protein ESM-1 according to the invention,compared to an immunodetection test of the state of the art, since asensitivity difference of between 5 and 10 fold can be observed betweenthese two tests, in favour of the immunodetection test of the invention.

Example 5 Comparative Results of Immunodetection Tests Using DifferentAntibodies of the Invention Directed Against the N-terminal Region ofProtein ESM-1 Produced by Transfected Cells of the Line HEK293

Immunodetection tests of the “sandwich” type were performed inaccordance with the protocol described in example 3.

In all cases, the antibody MEP14 was immobilized in the wells of a96-well plate of the ELISA type. After incubation of the plate thusprepared with solutions of known concentrations of recombinant ESM-1produced by transfected cells of the line HEK293 and washing, theantigen/antibody complex formed was incubated in the presence of asecond antibody, respectively the antibody MEC2, the antibody MEC15 andthe antibody MEC36.

The results are given in FIG. 2.

The results show that the use of the monoclonal antibodies directedagainst the N-terminal region of recombinant protein ESM-1 produced byeukaryotic cells in an immunoenzymatic test of the “sandwich” type leadsto a determination of protein ESM-1 of very high sensitivity, and thusthe sensitivity is reproducible from one antibody to another.

For the group of antibodies MEC15, MEC2 and MEC36 tested, concentrationsof the order of 0.15 to 0.2 nanograms per ml of ESM-1 could be detected.

Example 6 Application of the Immuno-detection Test According to theInvention to Quantifier Circulating Protein ESM-1 in Patients Sufferingfrom Sepsis of Different Levels of Severity

A. Materials and Methods.

A.1 Immuno-detection Test

The immunodetection test was performed in accordance with the protocoldescribed in example 3 above.

A.2. Test of Other Blood Markers

The quantitative test of serum soluble ICAM-1 protein was performed witha commercial ELISA test (Diaclone Research, Besangon, France). The platewas read using a spectrophotometer (Anthos Labtec LP40. France) at awavelength of 450 nanometers. The concentration of serum soluble ICAM-1was calculated from the measurements of optical density and expressed innanograms per ml, The normal value of the concentration of solubleICAM-1 protein was 571±168 nanograms per ml (219-1042; n=77).

The test of quantification of the protein procalcitonin was performedwith an immunological test of the ILMA type (Lumitest,B.R.A.M.S.-Diagnostica GmbH, Germany).

The normal value of procalcitonin is lower than 0.5 nanograms per ml.The values measured for patients suffering from Systemic InflammatoryResponse Syndrome (SIRS) were generally between 0.5 and 2 nanograms perml.

The quantity of C-reactive protein was measured by immuno-nephelometry.The normal value was lower than 10 mg/l (Morley et al., 1982, Ann. N.Y.Acad. Sci., vol. 389:406-418).

A.3 Subjects

The study was performed with four groups of subjects, one group ofnon-atopic healthy subjects (sex ratio 1) and three groups of patientssuffering from systemic and/or pulmonary septic inflammatory problems(sex ratio 1.6, age: 56±2 years).

All the non-atopic healthy subjects showed a negative response to theimmediate hypersensitivity cutaneous reaction test (prick test), andshowed no clinical history of allergies.

None of the 68 patients was treated with corticosteroids or hadundergone hemodialysis when blood samples were taken.

The experimental protocol was approved by the Local Ethics Committee ofthe Swiss University Hospitals.

A.4 Definitions.

The following terms have been used in this study.

Infection is a microbial phenomenon characterized by an inflammatoryresponse in the presence of micro-organisms or on the invasion ofnormally sterile tissue of the host by these microorganisms.

Systemic Inflammatory Response Syndrome (SIRS) with no sign ofinfection, is characterized by the presence of at least two of the fourfollowing clinical criteria:

a) fever or hypothermia (temperature above 100.4° F. [<38° C.] or >96.8°F.[>36° C.]);

b) tachycardia (>90 beats per minute);

c) tachypnea (>20 breaths per minute or PaCO₂<4.3 kPa[32 mm Hg]); and

d) an abnormal white blood cell count >12.000 cells/mm³; <4000cells/mm³, or the presence of more than 10% of immature formsrespectively.

Sepsis is defined as SIRS accompanied by an infection.

Severe sepsis is defined as a sepsis associated with a malfunction, ahypoperfusion or a hypotension of an organ, Abnormalities inhypoperfusion and perfusion may include, but are not limited to,oliguria (volume excreted <30 ml/hr), lactic acidosis (level of serumlactate greater than 2 mmol/L) or an acute deterioration of the mentalstate without sedation (reduction of at least 3 points with respect tothe base value according to the coma score of the Glasgow type).

Septic shock is defined as the presence of a sepsis accompanied by alasting reduction in the systolic blood pressure (<90 mmHg, or areduction of 40 mm Hg compared to the base value of the systolic bloodpressure), associated with the presence of perfusion abnormalities (seeabove), despite appropriate resuscitation and the need for vasoactiveamines to maintain adequate blood pressure (Crit. Care Med. 1992, vol.20:864-874).

A.5 Description of the Study

For each patient, the following biological and clinical parameters wereobtained: age, sex, principal and secondary diagnosis as defined inCrit. Care ed., 1992, vol. 20: 864-874, antecedents, treatment, vitalstate 10 days before blood sampling, serum and plasma concentrations ofprotein ESM-1.

For most of the patients, other blood biological parameters were alsomeasured such as circulating soluble ICAM-1 (sICAM-1), procalcitonin(proCT), and C-reactive protein (CRP).

Blood samples were taken from each of the patients (or healthyvolunteers) in dry tubes and in tubes treated with EDTA, the centrifugedfor 30 mn at a speed of 3500 r.p.m. in a centrifuge of type jouan C3I(France).

The serum and plasma samples were stored for a short time at −20° C.before the tests were performed.

The survival period of 10 days hospitalization was chosen by postulatingthat the infection had directly contributed to death in these patientsor that the contribution of the initial inflammatory problem compared toother causes of mortality could not be excluded.

A.7 Statistical Analysis

The data are expressed as the mean plus or minus the standard deviation.

The comparisons of mean levels of ESM-1, sICAM-1, procalcitonin and CRPbetween the different groups were performed using the “One way Analysis”test Anova (Bonferroni-Dunn), the Kruskal-Wallis test and theMann-Whitney test.

The correlation between the values of the markers was performed usingthe Spearman Rank test.

The correlation between the level of circulating marker and the severityof the patient's situation was performed using the “One Way” test ANOVA(Bonferroni-Dunn), the Mann-Whitney test and the Wilcoxon test.

In general, a value of p<0.05 was considered as statisticallysignificant.

The graphs were produced in the form of graphs in vertical barsrepresented by their median and the first and third quartiles.

B. Results

B.1 Correlation Between the Plasma and Serum Levels of ESM-1.

In a first test, it was determined if the conditions of taking the bloodsamples could affect the value of the ESM-1 level. The comparison of thevalues of the plasma and serum levels of ESM-1 in 41 healthy subjectshad shown a strong correlation (r=0.85; p<0.0001). In FIG. 3, it can beobserved that, as for the control group, no significant difference canbe detected for a given patient between the plasma and serum levels ofESM-1, for the first 36 patients of the study.

These results suggest for a start that the coagulation of the blood doesnot alter the ESM-1 level. These results also suggest that protein ESM-1may be quantified either from blood or plasma samples.

Therefore, only the serum level of ESM-1 was used for the rest of thestudy.

B.2 Control Group

A mean value of the serum level of ESM-1 was 0.7±0.4 nanograms per ml inhealthy subjects, as shown in table 1, while the serum level of ESM-1was 1.0+/−0.1 nanograms per ml in the group of atopic subjects.

Although the difference was small, it was statistically significant(p<0.05).

In consequence, the control group referred to in the study is the groupof healthy non-atopic subjects representative of the general population,and the mean value of the ESM-1 level in this group of healthynon-atopic subjects was determined to be the standard normal value.

B.3. Groups of Patients

The results of the circulating markers in the groups of patients aresummarized in table 1 and illustrated in FIGS. 4 and 5.

In the serum of patients admitted for severe sepsis, the levels sESM1,sICAM-1, CRP and proCT were strongly increased for each group ofpatients, compared to the control group (table 1).

These increased levels were statistically significant (p<0.01 or less)for all the markers and for all the groups of patients, with theexception of procalcitonin in the group of patients suffering fromsepsis (table 1, FIGS. 4 and 5).

The severity of the disorder was defined according to three levelsdesignated respectively sepsis, severe sepsis and septic shock. In eachof these groups, the ESM-1 levels increased in a way statisticallycorrelated with the severity of the disorder (p<0.01, n=61) (see table2).

Similar increases were found for the serum procalcitonin and CRP levels(respectively p=0.0001, n=34 and p<0.01, n=43).

However, the level of sICAM-1 increased to about 5 times the normalvalue, but showed a similar level in each of the groups of patients.

The CRP (Anova and Mann-Whitney) and ProCT levels (Mann-Whitney) enabledthe groups of patients suffering from sepsis to be distinguished fromthe patients suffering from severe sepsis, which was not the case forthe ESM-1 levels.

The ESM-1 (Anova and Mann-Whitney) and ProCT levels (Mann-Whitney)enabled the groups of patients suffering from severe sepsis to bedistinguished from the patients suffering from septic shock, which wasnot the case for the CRP levels (see table 2).

In view of these results, the CRP seems to be the most sensitive markerfor the least severe disorder and the ESM-1 level seems to be a bettermarker then the ProCT marker for the more severe sepsis.

A search for correlation between the serum levels of ESM-1, sICAM-1, CRPor ProCT established a significant correlation between the CRP andprocalcitonin levels (p<0.01, table 3).

However, no correlation was found between the other markers (see table3).

In addition, no correlation was observed between the levels of ESM-1 andof plasma creatinine in the group of patients suffering from septicshock (results not shown).

In order to determine if the serum levels of ESM-1 could predict a vitalprognosis, a first approach was to compare the mean levels of serumESM-1 between the patients having died and the patients having survived.

When all the patients were taken into account from admission forresuscitation and according to a fatal outcome or not, the resultsshowed that the level of serum ESM-1 at the day of hospitalization (day0) was significantly greater in the group of patients having died at day10 (day 10; p<0.01; FIG. 6A and FIG. 7A). It may be emphasized that suchdifferences were not shown with the other markers sICAM-1, CRP or ProCT(FIGS. 6B-9).

In a second approach, the level is of sESM-1 and s-ICAM1 were measuredboth on the day of hospitalization (day 0) and 24 hours after (day 1).The level of ESM-1 at day 0 was significantly higher in patients havingdied than in patents having survived (FIG. 7A). This differencepersisted at day 1 (n=20. p<0.05) (FIG. 7A).

Such a difference could not be observed with any of the other markerssICAM-1 (FIG. 7B) nor with CRP, procalcitonin or sICAM-1.

In the group of patients suffering from septic shock only, the serumlevels of ESM-1 at day 0 and at day 1 (n=11) were significantly higherin patients having died than in patients having survived (FIGS. 8A-B).

In each group separately, no statistically significant correlation wasfound between the levels of sICAM-1 at day 0. of sICAM-1 at day 1 (FIG.8B), of CRP at day O, or of ProCT at day 0 and the survival at day 10 ofeach patient.

The results presented in this example show that the mean value of theconcentration of circulating protein ESM-1 is greatly increased comparedto healthy subjects. This increase of the mean concentration ofcirculating ESM-1 is correlated with the level of clinical severity ofthe disorder. The highest levels of circulating ESM-1 at the earlystages of the disorder were associated with a lower probability ofsurvival of the patient after 10 days.

In agreement with earlier studies, the circulating markers, such as Creactive protein or CRP (Morley et al.), procalcitonin or ProCT (ASSICOTM et al.); (MULLER B et al.), (WANNER G A et al.), and soluble ICAM-1protein or sICAM-1 (KAVAL S et al.), (SESSLER C N et al. (1995) SESSLERC N et al. (1993) were also significantly increased in the patients ofthe study. The CRP and ProCT markers were correlated with the clinicalseverity of the patients.

However, the determination of the levels of the three markers CRP, ProCTand sICAM-1 did not allow prediction of vital prognosis, in contrast tothe determination of the level of circulating protein ESM-1. Inaddition, the predictive value of the serum level of ESM-1 waspredominantly linked to the initial value of serum ESM-1 at day 0 ofhospitalization.

In this study, the increase of the level of serum ESM-1 did notcorrelate with renal malfunction, as measured by the increase of plasmacreatinine level. This result suggests that the increased levels ofprotein ESM-1 in the serum in patents suffering from septic shock arisefrom deteriorated pulmonary tissue or from cells of the vascular wallsin a stressed state.

In conclusion, the results of the study presented in this example showthat ESM-1 represents a new marker of malfunction of the endothelialcells in patients suffering from sepsis.

In combination with values of other parameters of circulating markerssuch as the soluble adhesion molecules, the cytokines, procalcitonin orother markers of specific malfunction of certain cells, the level ofprotein ESM-1 supplies useful information on the clinical severity andoutcome for patients suffering from sepsis.

TABLE 1 Values of the concentration of circulating protein ESM-1 inhealthy volunteers and in different groups of patients suffering fromsepsis Blood markers sESM-1 sICAM-14 CRP Procalcitonin Group n (ng/ml)(ng/ml) (mg/l) (ng/ml) Normal value 20 0.7 ± 0.1.   571 ± 168  <10 <0.5(healthy non atopic) All patients 61 5.4 ± 0.8*** 2510 ± 261*** 194 ±21*** 39.8 ± 12.4*** Sepsis 29 2.5 ± 0.3*** 2336 ± 406*** 126 ± 15***2.0 ± 1.4   (pulmonary) Severe 12 4.5 ± 1.4*** 2696 ± 721*  292 ± 73** 23.8 ± 9.2***  sepsis Septic shock 20 10.0 ± 1.8***  2661 ± 373*** 245 ±29*** 90.8 ± 29.5***The values of the blood markers are expressed as the mean value±standarddeviation. Significant difference between each group of patients and thenormal value (group of healthy non-atopic subjects): *p<0.01;**p<0.0001; ***p<0.0001.

TABLE 2 Potential distinction of the severity of the state of thepatient as a function of the level of the circulating markers sESM-1sICAM-1 CRP ProCT Markers showing a significant difference (ANOVA) ECSand S/S p < 0.01 NS NS NS ECS and S p < 0.0001 NS p < 0.05 p < 0.01 S/Sand S NS NS p < 0.01 NS Kruskal-Wallis p < 0.01 NS p < 0.01 p = 0.0001Markers showing a significant difference (Mann-Whitney) ECS et-S/S p <0.05 NS NS p < 0.05 ECS and S p < 0.001 NS p < 0.01 p = 0.0001 S/S and SNS NS p < 0.05 p < 0.01 NS = non significant.

TABLE 3 Correlation between the different values of the blood markersPlasma Marker sESM-1 pESM-1 Age 1ICAM-1 CRP ProCT creatinine sESM-1 — p< 0.0001 NS NS NS NS NS sICAM-1 NS — — — NS NS — CRP NS — — NS — p <0.01 — ProCT NS — — NS p < 0.01 — — NS = non significant.

REFERENCES

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1. Hybridoma line MEC15 deposited at CNCM on 17 Oct. 2000 underaccession number I-2572.
 2. A monoclonal antibody produced by hybridomaline MEC15 deposited on 17 Oct. 2000 at the CNCM under accession numberI-2572.
 3. A kit for detecting protein ESM-1 in a sample, comprising: a)a first antibody comprising a monoclonal antibody produced by hybridomaline MEC 15 deposited at Collection de Cultures de Micro-organismes(CNCM) of Institut Pasteur on 17 Oct. 2000 under accession numberI-2572; and b) a second antibody specifically binding to a C-terminalregion of protein ESM-1 contained between amino acid in position 79 andamino acid in position 184 of SEQ ID NO:
 1. 4. The kit for detectionaccording to claim 3, wherein the second antibody is chosen from amongantibodies able to recognize an antigenic determinant selected from thegroup consisting of: a) the antigenic determinant AgD1 running fromproline in position 79 to cysteine in position 99 of SEQ ID NO: 1, b)the antigenic determinant AgD2 running from serine in position 119 tovaline in position 139 of SEQ ID NO: 1; and c) the antigenic determinantAgD3 running from glycine in position 159 to arginine in position 184 ofSEQ ID NO:
 1. 5. The kit for detection according to claim 4, wherein thesecond antibody comprises a monoclonal antibody selected from the groupconsisting of: a) the monoclonal antibody MEP 21 produced by a hybridomaline deposited at CNCM on 19 Nov. 1997 under accession number I-1944; b)the monoclonal antibody MEP08 produced by a hybridoma line deposited on19 Nov. 1997 at CNCM under accession number I-1941; c) the monoclonalantibody MEP14 produced by a hybridoma line deposited on 19 Nov. 1997 atCNCM under accession number I-1942; and d) the monoclonal antibody MEP19produced by a hybridoma line deposited on 19 Nov. 1997 at CNCM underaccession number I-1943.
 6. The kit for detection according to claim 3,wherein at least one of the two antibodies is covalently linked to amolecule enabling its direct or indirect detection.
 7. The kit fordetection according to claim 3, wherein the first or the second antibodyis immobilized on a support.
 8. The kit for detection according to claim7, wherein the antibody immobilized on a support is the antibodyspecifically recognizing the C-terminal region of protein ESM-1.
 9. Amethod for detecting protein ESM-1 in a sample comprising the followingsteps: a) placing a sample to be tested in contact with a firstantibody, wherein the first antibody is selected from: (i) an antibodyspecifically binding to a N-terminal region of protein ESM-1 containedbetween amino acid in position 20 and amino acid in position 78 of SEQID NO: 1 comprising a monoclonal antibody produced by hybridoma line MEC15 deposited at Collection de Cultures de Micro-organismes (CNCM) ofInstitut Pasteur on 17 Oct. 2000 under accession number 1-2572, or (ii)an antibody specifically binding to a C-terminal region of protein ESM-1contained between amino acid in position 79 and amino acid in position184 of SEQ ID NO: 1; b) placing a complex potentially formed between theprotein ESM-1 present in the sample and the first antibody in contactwith a second antibody, wherein the second antibody is selected from (i)or (ii) and binds to the region of ESM-1 not recognized by the firstantibody; and c) detecting the complex formed between protein ESM-1 andthe second antibody.
 10. The method of detection according to claim 9,wherein the first antibody is immobilized on a support.
 11. The methodof detection according to claim 9, wherein the first antibody is anantibody specifically binding to the C-terminal region of protein ESM-1and the second antibody is a monoclonal antibody produced by hybridomaline MEC 15 deposited at Collection de Cultures de Micro-organismes(CNCM) of Institut Pasteur on 17 Oct. 2000 under accession numberI-2572.
 12. The method of detection according to claim 9, wherein stepc) comprises detecting the complex with a biotinylated antibody able tofix to the second antibody.
 13. A method of quantifying protein ESM-1 invitro in a sample comprising measuring protein ESM-1 in the sample withthe kit of claim
 3. 14. A method for detecting elevated levels ofprotein ESM-1 in a plasma or serum sample of a human as indicative ofsepsis or septic shock in the human, comprising detecting protein ESM-1in the sample from the human with the kit of claim 3, determining aplasma or serum concentration of protein ESM-1 in the sample, andcomparing the plasma or serum concentration of protein ESM-1 to an ESM-1concentration indicative of lack of sepsis or septic shock to determineif the ESM-1 concentration is elevated and indicative of sepsis orseptic shock in the human.
 15. The method of claim 14, wherein theelevated levels of protein ESM-1 indicative of sepsis or septic shockcomprise at least 2.5 ng ESM-1 per mL of plasma or serum.
 16. The methodof claim 14, wherein the elevated levels of protein ESM-1 indicative ofsepsis or septic shock comprise at least 4.5 ng ESM-1 per mL of plasmaor serum.
 17. The method of claim 14, wherein the elevated levels ofprotein ESM-1 indicative of sepsis or septic shock comprise at least10.0 ng ESM-1 per mL of plasma or serum.